Stabilized soluble pre-fusion RSV F polypeptides

Abstract

Described are stable pre-fusion respiratory syncitial virus (RSV) F polypeptides, immunogenic compositions comprising the polypeptides and uses thereof for the prevention and/or treatment of RSV infection.

The invention claimed is: 
     
       1. A recombinant pre-fusion respiratory syncytial virus (RSV) Fusion (F) mutant polypeptide, comprising
 a mutation of the amino acid residue N/T at position 67 into I and/or a mutation of the amino acid residue S at position 215 into P, wherein the amino acid positions are given in reference to the sequence of RSV F protein from the A2 strain (SEQ ID NO: 1). 
 
     
     
       2. The pre-fusion RSV F mutant polypeptide according to  claim 1 , wherein the mutant polypeptide comprises an F1 domain and an F2 domain, and a linking sequence comprising from 1 to 10 amino acid residues, linking said F1 domain to said F2 domain. 
     
     
       3. The pre-fusion RSV F mutant polypeptide of  claim 1 , comprising a truncated F1 domain and an F2 domain, and a linking sequence comprising 1 to 10 amino acid residues, linking said F1 domain to said F2 domain. 
     
     
       4. The pre-fusion RSV F mutant polypeptide according to  claim 3 , wherein the mutant polypeptide comprises a heterologous trimerization domain linked to said truncated F1 domain. 
     
     
       5. The pre-fusion RSV F mutant polypeptide of  claim 1 , wherein the mutant polypeptide comprises at least one further mutation, wherein said mutation is selected from the group consisting of:
 (a) a mutation of the amino acid residue on position 46; 
 (b) a mutation of the amino acid residue on position 77; 
 (c) a mutation of the amino acid residue on position 80; 
 (d) a mutation of the amino acid residue on position 92; 
 (e) a mutation of the amino acid residue on position 175; 
 (f) a mutation of the amino acid residue on position 184; 
 (g) a mutation of the amino acid residue on position 185; 
 (h) a mutation of the amino acid residue on position 201; 
 (i) a mutation of the amino acid residue on position 209; 
 (j) a mutation of the amino acid residue on position 421; 
 (k) a mutation of the amino acid residue on position 426; 
 (l) a mutation of the amino acid residue on position 465; 
 (m) a mutation of the amino acid residue on position 486; 
 (n) a mutation of the amino acid residue on position 487; and 
 (o) a mutation of the amino acid residue on position 508. 
 
     
     
       6. The pre-fusion RSV F mutant polypeptide according to  claim 5 , wherein the at least one further mutation is selected from the group consisting of:
 (a) a mutation of the amino acid residue S on position 46 into G; 
 (b) a mutation of the amino acid residue K on position 77 into E; 
 (c) a mutation of the amino acid residue K on position 80 into E; 
 (d) a mutation of the amino acid residue E on position 92 into D; 
 (e) a mutation of the amino acid residue N on position 175 into P; 
 (f) a mutation of the amino acid residue G on position 184 into N; 
 (g) a mutation of the amino acid residue V on position 185 into N; 
 (h) a mutation of the amino acid residue K on position 201 into Q; 
 (i) a mutation of the amino acid residue K on position 209 into Q; 
 (j) a mutation of the amino acid residue K on position 421 into N; 
 (k) a mutation of the amino acid residue N on position 426 into S; 
 (l) a mutation of the amino acid residue K on position 465 into E or Q; 
 (m) a mutation of the amino acid residue D on position 486 into N; 
 (n) a mutation of the amino acid residue E on position 487 into Q, N or I; and 
 (o) a mutation of the amino acid residue K on position 508 into E. 
 
     
     
       7. The pre-fusion RSV F mutant polypeptide according to  claim 1 , wherein the mutant polypeptide comprises at least two mutations. 
     
     
       8. The pre-fusion RSV F mutant polypeptide of  claim 1 , wherein the heterologous trimerization domain comprises SEQ ID NO:3. 
     
     
       9. The pre-fusion RSV F mutant polypeptide according to  claim 8 , wherein the trimerization domain is linked to amino acid residue 495 of the RSV F protein. 
     
     
       10. The pre-fusion RSV F mutant polypeptide of  claim 1 , wherein the heterologous trimerization domain comprises SEQ ID NO:4. 
     
     
       11. The pre-fusion RSV F mutant polypeptide according to  claim 10 , wherein the trimerization domain is linked to amino acid residue 513 of the RSV F protein. 
     
     
       12. The pre-fusion RSV F mutant polypeptide of  claim 1 , wherein the linker between the F1 and the F2 domain comprises 5 amino acid residues. 
     
     
       13. The pre-fusion RSV F mutant polypeptide of  claim 12 , wherein the linker comprises SEQ ID NO:5. 
     
     
       14. The pre-fusion RSV F mutant polypeptide of  claim 1 , wherein the F1 domain and/or the F2 domain are from an RSV A strain. 
     
     
       15. The pre-fusion RSV F mutant polypeptide of  claim 1 , wherein the F1 domain and/or the F2 domain are from an RSV B strain. 
     
     
       16. The pre-fusion RSV F mutant polypeptide of  claim 1 , wherein the mutant polypeptide comprises a peptide selected from the group consisting of SEQ ID NO: 21-SEQ ID NO: 52 and SEQ ID NOs:71-89. 
     
     
       17. The pre-fusion RSV F mutant polypeptide of  claim 1 , wherein the mutant polypeptide does not comprise a HIS-Tag. 
     
     
       18. A nucleic acid molecule encoding the pre-fusion RSV F mutant polypeptide of  claim 1 . 
     
     
       19. The nucleic acid molecule of  claim 18 , wherein the nucleic acid molecule is codon-optimized for expression in mammalian cells. 
     
     
       20. A vector comprising the nucleic acid molecule of  claim 18 . 
     
     
       21. A composition comprising:
 the pre-fusion RSV F mutant polypeptide of  claim 1 , and a pharmaceutically acceptable carrier or excipient. 
 
     
     
       22. A method of inducing an immune response against RSV F protein in a subject, the method comprising:
 administering to the subject the pre-fusion RSV F mutant polypeptide of  claim 1  in an amount to induce an immune response against RSV F protein in the subject. 
 
     
     
       23. A vaccine comprising:
 the pre-fusion RSV F mutant polypeptide of  claim 1 . 
 
     
     
       24. A method of prophylaxing and/or treating RSV infection in a subject, the method comprising:
 administering to the subject the pre-fusion RSV F mutant polypeptide of  claim 1 , to prophylax against and/or treat the subject for RSV infection. 
 
     
     
       25. A vector comprising the nucleic acid molecule of  claim 19 . 
     
     
       26. The pre-fusion RSV F mutant polypeptide according to  claim 1 , comprising the mutation of the amino acid residue N/T at position 67 into I and the mutation of the amino acid residue S at position 215 into P. 
     
     
       27. The pre-fusion RSV F mutant polypeptide according to  claim 26 , further comprising a mutation of the amino acid residue K at position 66 into E, a mutation of the amino acid residue I at position 76 into V, and a mutation of the amino acid residue D at position 486 into N. 
     
     
       28. A nucleic acid molecule encoding the pre-fusion RSV F mutant polypeptide of  claim 26 . 
     
     
       29. A nucleic acid molecule encoding the pre-fusion RSV F mutant polypeptide of  claim 27 . 
     
     
       30. A vector comprising the nucleic acid molecule of  claim 28 . 
     
     
       31. A vector comprising the nucleic acid molecule of  claim 29 . 
     
     
       32. A vaccine comprising the pre-fusion RSV F mutant polypeptide of  claim 27  and/or a vector comprising a nucleic acid molecule encoding the same. 
     
     
       33. A method of prophylaxing and/or treating RSV infection in a subject, the method comprising:
 administering to the subject the pre-fusion RSV F mutant polypeptide of  claim 27  and/or a vector comprising a nucleic acid molecule encoding the same, to prophylax against and/or treat the subject for RSV infection. 
 
     
     
       34. The pre-fusion RSV F mutant polypeptide according to  claim 26 , further comprising a mutation of the amino acid residue S at position 46 into G, a mutation of the amino acid residue K at position 66 into E, and a mutation of the amino acid residue I at position 76 into V. 
     
     
       35. A nucleic acid molecule encoding the pre-fusion RSV F mutant polypeptide of  claim 34 . 
     
     
       36. A vector comprising the nucleic acid molecule of  claim 35 . 
     
     
       37. A vaccine comprising the pre-fusion RSV F mutant polypeptide of  claim 34  and/or a vector comprising a nucleic acid molecule encoding the same. 
     
     
       38. A method of prophylaxing and/or treating RSV infection in a subject, the method comprising:
 administering to the subject the pre-fusion RSV F mutant polypeptide of  claim 34  and/or a vector comprising a nucleic acid molecule encoding the same, to prophylax against and/or treat the subject for RSV infection.